Zaitsu and Ohkura, Analytical Biochemistry, 109, 109-113 (1980) describe the use of phenolic compounds as fluorogenic substrates for the horseradish-peroxidase (HRP)-mediated reaction with hydrogen peroxide in order to assay for peroxidase activity or the peroxide. Of the fluorogenic substrates, 3-(p-hydroxyphenyl)propionic acid (HPPA) was identified as being preferred in providing a rapid and sensitive assay. Tuuminen, et al., Journal of Immunoassay, 12(1), 29-46 (1991) recognized the observations of Zaitsu and Ohkura and applied the use of HPPA as a fluorogenic substrate of labelled HRP in an immunoassay.
A limitation accompanying the use of HPPA as a fluorogenic substrate for peroxidases used as labelling enzymes in enzyme immunoassay methods was recognized in Japanese Patent Application No. 4-234998 filed on Dec. 27, 1990, by Kohusai Shiyaku K. K. It was observed that peroxidase-mediated enzyme immunoassay methods using HPPA were intrinsically highly sensitive, but that HPPA formulated in buffers to provide the substrate solution undergoes condensation as a result of the presence of metal ions and is converted into a fluorescent substance which results in a rise in the reagent blank, thus decreasing sensitivity and measurement precision. The Japanese patent application discloses that a chelating agent such as an aminopolycarboxylic acid or aminopolyphosphonic acid, or salts thereof, can be used to stabilize formulated HPPA substrate solutions by reducing the rise in the reagent blank.
While demonstrating an improvement in the assay, following the technique illustrated in the Japanese application and other cited literature does not provide for an optimum assay. There is still room for improvement in enhancing the sensitivity of the assay and, in particular, in enhancing its utility by extending the effective working range of the assay by broadening the effective peroxidase reaction response curve, as defined by peroxidase concentration, time, or temperature. With conventional substrates containing a fluorogenic phenolic compound, hydrogen peroxide, and, preferably a metal chelator, there is a heretofor unrecognized limitation on these substrates that restricts their effective working range. The limitation is the formation of certain nonproductive product(s) during the course of the reaction of the substrate solution with peroxidase along with the productive formation of the fluorescent product of the reaction. The nonproductive product(s) possess optical characteristics interfering with the quantitative measurement of fluorescence generated by peroxidase activity. These nonproductive product(s) absorb light within the phenolic compound's fluorescent product excitation/emission spectra. Accordingly, the fluorescence measured is less than that which the reaction generates, the difference being that absorbed by nonproductive product(s).
Having the above in mind, the presence of the nonproductive product(s) limits the effective working range of available fluorogenic phenolic substrates for determining peroxidase activity. In the assays, there is a point, either determined by reaction time, or peroxidase concentration, or temperature, where the concentration of nonproductive products(s) increases to a critical point where the continued rise in fluorescence is defeated, and indeed the fluorescence may be observed to decrease, thus disabling the ability to distinguish peroxidase concentrations or reaction times from dissimilar values. In other words, due to the presence of the nonproductive product(s), the measured fluorescence at one concentration is depressed to the same as the measured fluorescence at a lower concentration or the measured fluorescence is depressed to the same as the measured fluorescence at lower times during the reaction. Therefore, it would be desirable to either fashion a formulation so that this critical point can be manipulated within an assay to increase effective working range, or to increase effective working range by providing means for minimization of the consequences of the critical point by eliminating the nonproductive product(s).